TCF responsive element

Abstract

Disclosed are DNA elements and constructs useful for obtaining tumour-selective gene expression in tumours having a mutated β-catenin/APC pathway. In particular, the use of these constructs to express genes encoding therapeutic proteins in colorectal cancer cells is described. The constructs comprise multiple repeats of a TCF-binding element operably linked to a promoter. By means of such a construct, tumour cell-specific expression of a prodrug-converting enzyme such as nitroreductase may be achieved. Coupled with systemic administration of a suitable prodrug, such as CB1954, selective killing of such tumour cells can be demonstrated.

What is claimed is:  
     
       1. A nucleic acid construct comprising: 
       1) a V-cell factor (TCF) response element comprising:  
       at least one TCF binding element having the sequence CTTTGNN wherein N is A or T; and  
       an operably linked promoter; and  
       2) an expressible gene that is useful for the treatment of a cancer that is characterised by deregulation of Writ pathway signalling; wherein the expressible gene is operably linked to the TCF response element which enables inducible expression of the gene.  
     
     
       2. The nucleic acid construct according to  claim 1  wherein the expressible gene is selected from the group consisting of: a gene encoding a toxin, a prodrug activating enzyme, or an immtmornodulatory agent; a tumor suppressor gene; and an apoptotic gene. 
     
     
       3. The nucleic acid construct of  claim 1  or  2  wherein the expressible gene encodes a toxin or a prodrug activating enzyme. 
     
     
       4. The nucleic acid construct of  claim 3  wherein the expressible gene encodes a nitroreductase capable of activating CB1954. 
     
     
       5. The nucleic acid construct of  claim 1  or  2  wherein the promoter is selected from the group consisting of an SV40 promoter, an E1B promoter, and a c-Fos promoter. 
     
     
       6. The nucleic acid construct of  claim 4 , wherein the promoter is an E1B promoter. 
     
     
       7. A nucleic acid construct comprising: 
       1) a TCF response element comprising:  
       at least 5 TCF binding elements having the sequence CTTTGNN wherein N is A or T; and  
       an operably linked promoter; and  
       2) an expressible gene that is useful for the treatment of a cancer that is characterised by deregulation of Wnt pathway signalling; wherein the expressible gene is operably linked to the TCF response element which enables inducible expression of the gene.  
     
     
       8. The nucleic acid construct of  claim 7  wherein the TCF response element comprises between 5 and 10 TCF binding elements. 
     
     
       9. The nucleic acid construct  claim 8  wherein the TCF response element comprises 5 TCF binding elements. 
     
     
       10. A nucleic acid construct comprising: 
       1) a TCF response element comprising:  
       at least two TCF binding elements having the sequence CTTTGNN wherein N is A or T; and  
       an operably linked promoter; and  
       2) an expressible gene that is useful for the treatment of a cancer that is characterised by deregulation of Wnt pathway signalling; wherein die expressible gene is operably linked to the TCF response element which enables inducible expression of the gene, and wherein the TCF binding elements are separated from each other by between 3 and 20 nucleotides.  
     
     
       11. The nucleic acid construct of  claim 10  wherein the TCF binding elements are separated from each other by between 3 and 12 nucleotides. 
     
     
       12. The nucleic acid construct of  claim 11  wherein the TCF binding elements are separated from each other by between 10 and 12 nucleotides. 
     
     
       13. A nucleic acid construct comprising: 
       1) a TOF response element comprising:  
       at least one TCF binding element having the sequence CTTTGNN wherein N is A or T; and  
       an operably linked promoter; and  
       2) an expressible gene that is useful for the treatment of a cancer that is characterised by deregulation of Wnt pathway signalling; wherein the expressible gene is operably linked to the TUE response element which enables inducible expression of the, and wherein the TCF binding element closest to the promoter is between 140 and 10 nucleotides from the TATA box of the promoter.  
     
     
       14. The nucleic acid construct of  claim 13  wherein the promoter comprises a TATA box, and the TCF binding element closest to the promoter is between 100 and 10 nucleotides from the TATA box of the promoter. 
     
     
       15. The nucleic acid construct of  claim 14  wherein the promoter comprises a TATA box, and the TOE binding element closest to the promoter is between 50 and 10 nucleotides from the TATA box of the promoter. 
     
     
       16. The nucleic acid construct of  claim 15  wherein the promoter comprises a TATA box, and the TCE binding element closest to the promoter is between 30 and 15 nucleotides from the TATA box of the promoter. 
     
     
       17. The nucleic acid construct of  claim 10  wherein the TOE binding elements are separated from each other by 3 or 4 nucleotides and wherein the promoter comprises a TATA box, and the TOE binding element closest to the promoter is 25 nucleotides from the TATA box of the promoter. 
     
     
       18. A method of treatment for cancer, comprising administering to a patient in need of such treatment an effective dose of the nucleic acid construct of any one of claims  1 ,  2 ,  7 ,  10 , or  13 . 
     
     
       19. A composition comprising the nucleic acid construct of any one of claims  1 ,  2 ,  7 ,  10 , or  13  and a pharmaceutically acceptable excipient. 
     
     
       20. The nucleic acid construct according to  claim 7 , wherein the expressible gene is selected from the group consisting of: a gene encoding a toxin, a prodrug activating enzyme, or an immunomodulatory agent; a tumor suppressor gene; and an apoptotic gene. 
     
     
       21. The nucleic acid construct according to  claim 10 , wherein the expressible gene is selected from the group consisting of: a gene encoding a toxin, a prodrug activating enzyme, or an immunomodulatory agent; a tumor suppressor gene; and an apoptotic gene. 
     
     
       22. The nucleic acid construct according to  claim 13 , wherein the expressible gene is selected from the group consisting of: a gene encoding a toxin, a prodrug activating enzyme, or an immunomodulatory agent; a tumor suppressor gene; and an apoptotic gene. 
     
     
       23. The nucleic acid construct of any one of claims  1 ,  2 ,  7 ,  10 ,  13 ,  20 ,  21 , or  22  wherein at least one TOE binding element has the nucleotide sequence CTTTGAT. 
     
     
       24. A vector comprising the nucleic acid construct of any one of claims  1 ,  2 ,  7 ,  10 ,  13 ,  20 ,  21 , or  22 . 
     
     
       25. A host cell transfected with the vector of  claim 24 . 
     
     
       26. A method of treatment for cancer, comprising administering to a patient in need of such treatment an effective dose of the vector of  claim 24 . 
     
     
       27. A method of treatment for cancer, comprising administering to a patient in need of such treatment an effective dose of the host cell of  claim 25 . 
     
     
       28. A composition comprising the vector of  claim 24 . 
     
     
       29. A composition comprising the host cell of  claim 25  and a pharmaceutically acceptable excipient.